This proposal outlines methods to study the enzymatic activity, the heterogeneity, and the copper content of copper-zinc superoxide dismutase (SOD) obtained from partly purified lysates of young and old bovine erythrocytes. The observations from the red cell systems will be compared to those obtained in the reaction of SOD with hydrogen peroxide in order to assess whether or not peroxide may be a causative agent for these changes. Superoxide dismutase from older bovine erythrocytes has been shown to lose activity and increase in electrophoretic heterogeneity and sensitivity to the inhibitors cyanide and DDC. The chemical events leading to these changes in the dismutase and their contribution to red cell aging have not been elucidated. This proposal addresses the possibility that the reaction of the dismutase with hydrogen peroxide may be physiologically relevant and lead to an increase in hetereogeneity and loss of activity which would contribute to the aging and destruction of the red blood cell. Standard methodology will be employed for determining superoxide dismutase activity. Heterogeneity of the SOD will be determined by electrophoresis staining for protein and activity. Methods of purification of SOD and its minor forms will be developed and the enzymatic activity, heterogeneity, and copper content will be studied in gels of partly purified lysates. Studies of the amino acid composition will be performed on purified forms from old and young cells as well as on enzyme reacted with peroxide. In order to monitor the copper content of the dismutase in partly purified cell lysates, a new method using diethyldithiocarbamate (DDC) to stain polyacrylamide gels will be employed. This new method for staining gels is described in this proposal and has worked well in studies of the purified copper-zinc superoxide dismutase.